Project Summary/Abstract The goal of this proposal is to bridge gaps in our understanding of how the HER2/HER3 heterocomplex is activated by performing direct enzymatic and structural studies on full-length HER2 and HER3 receptors. EGFR, HER2, HER3, and HER4 comprise the Human Epidermal Growth Factor Receptor (HER) family of receptor tyrosine kinases, which are indispensable for cellular homeostasis. These receptors relay extracellular signals into intracellular responses through ligand-dependent homo- or hetero-dimerization resulting in the activation of their intracellular kinase domains. HER receptor dysfunction contributes to the pathogenesis of a broad spectrum of human cancers. Activation of the HER2/HER3 heterocomplex in particular is known to correlate with aggressive tumor phenotypes. Recently, the first cancer-related somatic mutations in HER3 were reported, further underscoring the importance of HER3 in driving tumorigenesis. Many anti-HER therapeutics developed through pharmacological scrutiny of HER receptor subdomains have had remarkable effects on improving disease outcomes. Yet, there are still subsets of patients that do not respond well, or at all, to such therapeutics despite the demonstrated contribution of HER receptor signaling to their disease. This suggests that there are aspects of the HER2/HER3 activation mechanism that cannot be efficiently interrogated by studies conducted solely on receptor fragments. The lack of direct investigations into full-length HER receptors ultimately limits progress towards novel therapeutic design. The specific goals of this proposal are to understand: (1) the oligomeric transitions underlying HER2/HER3 complex activation (2) the effect of cancer mutations in the HER3 extracellular domain on the canonical mechanism of ligand-dependent HER2/HER3 complex activation, and (3) the structural basis of HER2/HER3 complex activation. The multidisciplinary approach described in this application relies on advances in membrane protein biochemistry and breakthroughs in electron cryo-microscopy to provide invaluable insights into the regulation of HER receptor activation. In light of the recent discovery of HER3 mutations that potentiate its oncogenic properties, such findings will establish a necessary framework for rational design of novel chemotherapeutics targeting the subset of patients carrying these mutations. The long-term goal of this work is to innovate personalized medicine for patients affected by HER-specific diseases and help improve their outcomes.